Nested polymerase chain reaction for detection of Ehrlichia equi genomic DNA in horses and ticks (Ixodes pacificus)

Jeffrey E. Barlough, John E Madigan, Elfriede DeRock, Luisa Bigornia

Research output: Contribution to journalArticle

127 Scopus citations

Abstract

A nested polymerase chain reaction for detecting Ehrlichia equi in horses and ticks (Ixodes pacificus) was developed. A major second-round PCR product of 928 bp could be readily visualized in ethidium bromide-stained agarose minigels. An internal probe was used to verify the identity of the amplified product by non-radioactive (digoxigenin-based) Southern blotting; additional confirmation was provided by DNA sequence analysis. A dilution study testing the sensitivity of the PCR indicated that DNA derived from ≤ 7.6 but > 3 infected neutrophils was sufficient to generate a PCR signal. The specificity of the PCR was examined using a panel of rickettsiae, of which only E. equi and the closely-related human granulocytotropic ehrlichia produced PCR bands. In an in vivo infection study, E. equi DNA was detected in blood buffy-coat cells of an experimentally-infected horse on days three through 14 post- inoculation. In a separate study, three of six adult I. pacificus that as nymphs had been fed on an experimentally infected horse were found to be PCR- positive for E. equi.

Original languageEnglish (US)
Pages (from-to)319-329
Number of pages11
JournalVeterinary Parasitology
Volume63
Issue number3-4
DOIs
StatePublished - Jun 1996

Keywords

  • Diagnosis Rickettsia
  • DNA sequence analysis
  • Ehrlichia equi
  • Horse
  • Ixodes pacificus
  • Polymerase chain reaction
  • Southern hybridization test

ASJC Scopus subject areas

  • Animal Science and Zoology
  • Parasitology
  • veterinary(all)

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